This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall aim of this research program is to determine how [NiFe]-hydrogenase enzymes are made in bacteria. These enzymes are found in many microorganisms, including Escherchia coli (E. coli) and the pathogenic bacteria Helicobacter pylori, and they are key components in the metabolic pathways that either make hydrogen gas or use it as an energy source. The enzyme has a complicated catalytic active site that contains nickel and iron bound to the protein in addition to several non-protein ligands. It is known that the biosynthesis of hydrogenases requires the coordinated activity of multiple helper proteins to deliver all of the components and assemble the metallocenter correctly. However, very little is known about the mechanisms of action of the individual proteins, how they interrelate with each other, how metal is transferred, or how metal selectivity is achieved. An understanding of this multi-step process is essential in order to realize the potential of hydrogenase enzymes for biotechnology and consumable energy applications, or to evaluate the component proteins as antibiotic targets. Furthermore, this study will contribute to our knowledge about intracellular transition metal homeostasis, a fundamental aspect of life.